Engineered primate L-methioninase for therapeutic purposes

ABSTRACT

Methods and compositions relating to the engineering of an improved protein with methionine-γ-lyase enzyme activity are described. For example, in certain aspects there may be disclosed a modified cystathionine-γ-lyase (CGL) comprising one or more amino acid substitutions and capable of degrading methionine. Furthermore, certain aspects of the invention provide compositions and methods for the treatment of cancer with methionine depletion using the disclosed proteins or nucleic acids.

The present application is a continuation of U.S. application Ser. No.15/297,102, filed Oct. 18, 2016, which is a divisional of U.S.application Ser. No. 14/472,750, filed Aug. 29, 2014, which claims thepriority benefit of U.S. provisional application No. 61/871,768, filedAug. 29, 2013, the entire contents of each of which are incorporatedherein by reference.

The invention was made with government support under Grant No. R01CA154754 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of medicine andbiology. More particularly, it concerns an improved humanmethionine-γ-lyase (hMGL) for use in the treatment of cancer.

2. Description of Related Art

The demand for the essential amino acid, L-methionine, is exceptionallyhigh in cancerous tissues. Depletion of methionine has been shown to beeffective in killing a wide variety of tumor types without adverselyaffecting non-cancerous tissues. Methionine depletion can be effectedvia the action of enzymes that hydrolyze the amino acid. While humanmethionine depleting enzymes did not previously exist, a bacterialenzyme from Pseudomonas aeruginosa, methionine-γ-lyase, was shown to betherapeutically effective in the clinic and had been evaluated inclinical trials. However, methionine-γ-lyase, being a bacterial protein,is highly immunogenic, eliciting the formation of specific antibodies,leading to adverse reactions and also reduced activity.Methionine-γ-lyase also has a very short half-life of only about 2 h invitro and in vivo, necessitating very frequent and impractically highdosing to achieve systemic depletion.

Systemic methionine depletion is the focus of much research and has thepotential to treat cancers, such as metastatic breast cancer, prostate,neuroblastoma, and pancreatic carcinoma among others. Although there ismuch excitement for this therapeutic approach, the bacterially-derivedmethionine-γ-lyase has serious shortcomings (immunogenicity and rapiddeactivation in serum, as discussed above) that greatly dampenenthusiasm for its use as a chemotherapeutic agent.

Previously, an engineered human methionine-γ-lyase (hMGL-NLV) wascreated by introducing three key amino acid substitutions in the humanenzyme cystathionine-γ-lyase (CGL): E59N, R119L, E339V. See, U.S. Pat.No. 8,709,407, which is incorporated herein by reference. Unlike nativeCGL, which displays essentially no catalytic activity towardsL-methionine, the E59N, R119L, E339V variant enzyme (hMGL-NLV) displaysrobust L-methionine degrading activity in vivo and in vitro.Nonetheless, there remains a need to develop human L-methioninedegrading enzymes with higher catalytic activity so that a therapeuticeffect can be attained with lower dosing and/or less frequentadministration of the enzyme.

SUMMARY OF THE INVENTION

Provided herein are human methionine-γ-lyase (hMGL) mutants with highercatalytic activity than hMGL-NLV. The preferred improved hMGL proteinsexhibit 6-10 fold higher activity. Due to the higher catalytic activity,the provided hMGL proteins may display higher therapeutic potency formethionine depletion and thus lower concentrations of therapeutic agentmay be required for dosing of patients. This enzyme was engineered byintroducing amino acid substitutions in the human enzymecystathionine-γ-lyase (CGL) at positions comprising residues E59, R119,or E339. In one particular embodiment, preferred substitutions of E59,R119, and E339 in hCGL include, respectively, L-asparagine (at position59), L-leucine (at position 119) and L-valine (at position 339). Thepresent invention discloses compositions of matter displaying highercatalytic activity relative to the hMGL-NLV variants. These containsubstitutions at least at two of the E59, R119, or E339 positionsidentified as critical for conferring L-methionine-γ-lyase activitycompared to the hMGL-NLV variants. The higher catalytic activity ofthese variants is important because lower concentrations of therapeuticagent may be required for dosing of patients.

The variants having additional amino acid substitutions include SEQ IDNO: 3, hCGL-E59N-S63L-L91M-R119L-K268R-T311G-E339V-I353S (hCGL-8mut-1);SEQ ID NO: 4, hCGL-E59I-S63L-L91M-R119L-K268R-T311G-E339V-I353S(hCGL-8mut-2); SEQ ID NO: 5,hCGL-E59N-S63L-L91M-R119A-K268R-T311G-E339V-I353S (hCGL-8mut-3); and SEQID NO: 6, hCGL-E591-S63L-L91M-R119A-K268R-T311G-E339V-I353S(hCGL-8mut-4).

Certain aspects of the present invention overcome a major deficiency inthe art by providing improved enzymes that comprise human polypeptidesequences having methionine-γ-lyase (MGL) activity, which may besuitable for cancer therapy and have low immunogenicity, improved serumstability, and improved catalytic activity. Accordingly, in a firstembodiment there is provided a modified polypeptide (i.e., enzyme),particularly an enzyme variant with methionine-degrading activityderived from primate enzymes related to MGL. For example, the novelenzyme variant may have an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 3-6. In particular, the variant may be derivedfrom human enzymes, such as human cystathionine-γ-lyase (CGL). Incertain aspects, there may be a polypeptide comprising a modified humanCGL capable of degrading methionine. In some embodiments, thepolypeptide may be capable of degrading methionine under physiologicalconditions. For example, the polypeptide may have a catalytic efficiencyfor methionine (k_(cat)/K_(M)) of at least or about 0.01, 0.05, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10⁴, 10⁵, 10⁶M⁻¹s⁻¹ or any range derivable therein. In further aspects, thepolypeptide may display a catalytic activity towards L-homocystine up tok_(cat)/K_(M) of 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40,35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6,0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001 M⁻¹s⁻¹ or any rangederivable therein.

A modified polypeptide as discussed above may be characterized as havinga certain percentage of identity as compared to an unmodifiedpolypeptide (e.g., a native polypeptide) or to any polypeptide sequencedisclosed herein. For example, the unmodified polypeptide may compriseat least or up to about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 350, 400 residues (or any range derivable therein) of anative primate cystathionase (i.e., cystathionine-γ-lyase). Thepercentage identity may be about, at most or at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or anyrange derivable therein) between the unmodified portions of a modifiedpolypeptide (i.e., the sequence of the modified polypeptide excludingany substitutions at amino acid positions 59, 63, 91, 119, 268, 311, 339and/or 353) and the native polypeptide. It is also contemplated that thepercent identity discussed above may relate to a particular modifiedregion of a polypeptide as compared to an unmodified region of apolypeptide. For instance, a polypeptide may contain a modified ormutant substrate recognition site of cystathionase that can becharacterized based on the identity of the amino acid sequence of themodified or mutant substrate recognition site of cystathionase to thatof an unmodified or mutant cystathionase from the same species or acrossthe species. A modified or mutant human polypeptide characterized, forexample, as having at least 90% identity to an unmodified cystathionasemeans that at least 90% of the amino acids in that modified or mutanthuman polypeptide are identical to the amino acids in the unmodifiedpolypeptide.

Such an unmodified polypeptide may be a native cystathionase,particularly a human isoform or other primate isoforms. For example, thenative human cystathionase may have the sequence of SEQ ID NO: 1.Non-limiting examples of other native primate cystathionases includePongo abelii cystathionase (Genbank ID: NP_001124635.1; SEQ ID NO: 7),Macaca fascicularis cystathionase (Genbank ID: AAW71993.1; SEQ ID NO:8), Pan troglodytes cystathionase (Genbank ID: XP_513486.2; SEQ ID NO:9), and Pan paniscus cystathionase (Genbank ID: XP_003830652.1; SEQ IDNO: 10). Exemplary native polypeptides include a sequence having about,at most or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% identity (or any range derivable therein) ofSEQ ID NOs: 1 or 7-10 or a fragment thereof. For example, the nativepolypeptide may comprise at least or up to about 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 415 residues (or anyrange derivable therein) of the sequence of SEQ ID NOs: 1 or 7-10.

In some embodiments, the native CGL may be modified by one or more othermodifications, such as chemical modifications, substitutions,insertions, deletions, and/or truncations. For example, themodifications may be at a substrate recognitions site of the nativeenzyme. In a particular embodiment, the native CGL may be modified bysubstitutions. For example, the number of substitutions may be four,five, six, seven, or more. In further embodiments, the native CGL may bemodified in the substrate recognition site or any location that mayaffect substrate specificity. For example, the modified polypeptide mayhave the at least one amino acid substitution at amino acid positionscorresponding to E59, S63, L91, R119, K268, T311, E339, and/or I353 ofSEQ ID NO: 1 or amino acid positions of 59, 63, 91, 119, 268, 311, 339,and/or 353 of a primate CGL. For example, the primate may be human,Pongo abelii, Macaca fascicularis, Pan troglodyte, or Pan paniscus.

In certain embodiments, the substitutions at amino acid positions 59,63, 91, 119, 268, 311, 339, and/or 353 is an aspartic acid (N), a valine(V), a leucine (L), a methionine (M), an arginine (R), a glycine (G), analanine (A), or a serine (S). In particular embodiments, themodification are one or more substitutions selected from the groupconsisting of E59N, E59I, S63L, L91M, R119L, R119A, K268R, T311G, E339V,and I353S. In a further embodiment, the substitutions may comprise aS63L, L91M, K268R, T311G, E339V, and I353S substitutions. In a stillfurther embodiment, the substitutions may comprise additionalsubstitutions of either E59N or E59I and either R119L or R119A.

In some embodiments, the native CGL may be a human CGL. In a particularembodiment, the substitutions are a combination of E59N, S63L, L91M,R119L, K268R, T311G, I353S, and E339V of human CGL (for example, themodified polypeptide having the amino acid sequence of SEQ ID NO: 3, afragment or homolog thereof), a combination of E59I, S63L, L91M, R119L,K268R, T311G, I353S, E339V of human CGL (for example, the modifiedpolypeptide having the amino acid sequence of SEQ ID NO: 4, a fragmentor homolog thereof), a combination of E59N, S63L, L91M, R119A, K268R,T311G, I353S, and E339V of human CGL (for example, the modifiedpolypeptide having the amino acid sequence of SEQ ID NO: 5, a fragmentor homolog thereof), or a combination of E59I, S63L, L91M, R119A, K268R,T311G, I353S, and E339V of human CGL (for example, the modifiedpolypeptide having the amino acid sequence of SEQ ID NO: 6, a fragmentor homolog thereof). In a further embodiment, the modified polypeptidemay be a Pongo abelii CGL-NLMLRGSV mutant (SEQ ID NO: 11), Pongo abeliiCGL-ILMLRGSV mutant (SEQ ID NO: 12), Pongo abelii CGL-NLMARGSV mutant(SEQ ID NO: 13), Pongo abelii CGL-ILMARGSV mutant (SEQ ID NO: 14),Macaca fascicularis CGL-NLMLRGSV mutant (SEQ ID NO: 15), Macacafascicularis CGL-ILMLRGSV mutant (SEQ ID NO: 16), Macaca fascicularisCGL-NLMARGSV mutant (SEQ ID NO: 17), Macaca fascicularis CGL-ILMARGSVmutant (SEQ ID NO: 18), Pan troglodytes CGL-NLMLRGSV mutant (SEQ ID NO:19), Pan troglodytes CGL-ILMLRGSV mutant (SEQ ID NO: 20), Pantroglodytes CGL-NLMARGSV mutant (SEQ ID NO: 21), Pan troglodytesCGL-ILMARGSV mutant (SEQ ID NO: 22), Pan paniscus CGL-NLMLRGSV mutant(SEQ ID NO: 23), Pan paniscus CGL-ILMLRGSV mutant (SEQ ID NO: 24), Panpaniscus CGL-NLMARGSV mutant (SEQ ID NO: 25), or Pan paniscusCGL-ILMARGSV mutant (SEQ ID NO: 26).

In some aspects, the present invention also contemplates polypeptidescomprising the modified CGL linked to a heterologous amino acidsequence. For example, the modified CGL may be linked to theheterologous amino acid sequence as a fusion protein. In a particularembodiment, the modified CGL may be linked to amino acid sequences, suchas an IgG Fc, albumin, an albumin binding peptide, or an XTENpolypeptide for increasing the in vivo half-life.

To increase serum stability, the modified CGL may be linked to one ormore polyether molecules. In a particular embodiment, the polyether maybe polyethylene glycol (PEG). The modified polypeptide may be linked toPEG via specific amino acid residues, such as lysine or cysteine. Fortherapeutic administration, such a polypeptide comprising the modifiedCGL may be dispersed in a pharmaceutically acceptable carrier.

In some aspects, a nucleic acid encoding such a modified CGL iscontemplated. In some embodiments, the nucleic acid has been codonoptimized for expression in bacteria. In particular embodiments, thebacteria is E. coli. In other aspects, the nucleic acid has been codonoptimized for expression in fungus (e.g., yeast), insects, or mammals.The present invention further contemplates vectors, such as expressionvectors, containing such nucleic acids. In particular embodiments, thenucleic acid encoding the modified CGL is operably linked to a promoter,including but not limited to heterologous promoters. In one embodiment,a modified CGL may be delivered to a target cell by a vector (e.g., agene therapy vector). Such viruses may have been modified by recombinantDNA technology to enable the expression of the modified CGL-encodingnucleic acid in the target cell. These vectors may be derived fromvectors of non-viral (e.g., plasmids) or viral (e.g., adenovirus,adeno-associated virus, retrovirus, lentivirus, herpes virus, orvaccinia virus) origin. Non-viral vectors are preferably complexed withagents to facilitate the entry of the DNA across the cellular membrane.Examples of such non-viral vector complexes include the formulation withpolycationic agents, which facilitate the condensation of the DNA andlipid-based delivery systems. An example of a lipid-based deliverysystem would include liposome based delivery of nucleic acids.

In still further aspects, the present invention further contemplateshost cells comprising such vectors. The host cells may be bacteria(e.g., E. coli), fungal cells (e.g., yeast), insect cells, or mammaliancells. To further differentiate desired CGL mutants with methioninedegrading activity from the native CGL, host cells having deletions ofilvA and metA (e.g., E. coli ilvA⁻metA⁻) may be prepared and used toidentify desired mutants.

In some embodiments, the vectors are introduced into host cells forexpressing the modified CGL. The proteins may be expressed in anysuitable manner. In one embodiment, the proteins are expressed in a hostcell such that the protein is glycosylated. In another embodiment, theproteins are expressed in a host cell such that the protein isaglycosylated.

Certain aspects of the present invention also contemplate methods oftreatment by the administration of the modified CGL peptide, the nucleicacid encoding the modified CGL in a gene therapy vector, or theformulation of the present invention, and in particular methods oftreating tumor cells or subjects with cancer. The subject may be anyanimal, such as a mouse. For example, the subject may be a mammal,particularly a primate, and more particularly a human patient. In someembodiments, the method may comprise selecting a patient with cancer. Incertain aspects, the subject or patient may be maintained on amethionine-restricted diet or a normal diet.

In some embodiments, the cancer is any cancer that is sensitive tomethionine depletion. In one embodiment, the present inventioncontemplates a method of treating a tumor cell or a cancer patientcomprising administering a formulation comprising such a polypeptide. Insome embodiments, the administration occurs under conditions such thatat least a portion of the cells of the cancer are killed. In anotherembodiment, the formulation comprises such a modified CGL withmethionine degrading activity at physiological conditions and furthercomprising an attached polyethylene glycol chain. In some embodiment,the formulation is a pharmaceutical formulation comprising any of theabove discussed CGL variants and pharmaceutically acceptable excipients.Such pharmaceutically acceptable excipients are well known to those ofskill in the art. All of the above CGL variants may be contemplated asuseful for human therapy.

In a further embodiment, there may also be provided a method of treatinga tumor cell comprising administering a formulation comprising anon-bacterial (mammalian, e.g., primate or mouse) modified CGL that hasmethionine degrading activity or a nucleic acid encoding thereof

Because tumor cells are dependent upon their nutrient medium formethionine, the administration or treatment may be directed to thenutrient source for the cells, and not necessarily the cells themselves.Therefore, in an in vivo application, treating a tumor cell includescontacting the nutrient medium for a population of tumor cells with theengineered methioninase. In this embodiment, the medium can be blood,lymphatic fluid, spinal fluid and the like bodily fluid where methioninedepletion is desired.

In accordance with certain aspects of the present invention, such aformulation containing the engineered methioninase can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intrasynovially, intratracheally, intranasally,intravitreally, intravaginally, intrarectally, intratumorally,intramuscularly, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularly, orally,topically, by inhalation, infusion, continuous infusion, localizedperfusion, via a catheter, via a lavage, in lipid compositions (e.g.,liposomes), or by other method or any combination of the forgoing aswould be known to one of ordinary skill in the art.

In a further embodiment, the method may also comprise administering atleast a second anticancer therapy to the subject. The second anticancertherapy may be a surgical therapy, chemotherapy, radiation therapy,cryotherapy, hormone therapy, immunotherapy or cytokine therapy.

In one embodiment, a composition comprising an engineered methioninaseor a nucleic acid encoding an engineered methioninase is provided foruse in the treatment of a tumor in a subject. In another embodiment, theuse of an engineered methioninase or a nucleic acid encoding anengineered methioninase in the manufacture of a medicament for thetreatment of a tumor is provided. Said engineered methioninase may beany engineered methioninase of the embodiments.

Embodiments discussed in the context of methods and/or compositions ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the terms “encode” or “encoding,” with reference to anucleic acid, are used to make the invention readily understandable bythe skilled artisan; however, these terms may be used interchangeablywith “comprise” or “comprising,” respectively.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1—Sequence alignment of hCGL with hCGL-8mut-1-4. Highlightedresidues indicate mutated residues and their position. hCGL=SEQ ID NO:1; hCGL-8mut-1=SEQ ID NO: 3; hCGL-8mut-2=SEQ ID NO: 4; hCGL-8mut-3=SEQID NO: 5; and hCGL-8mut-4=SEQ ID NO: 6.

FIG. 2—The effect on prostate tumor cell lines PC3 (open circle) andDU145 (solid square) treated with titrations of hCGL-8mut-1 resultingwith apparent IC₅₀ values of 0.25 μM and 0.21 μM, respectively.

FIG. 3—Activity of PEGylated hCGL-8mut-1 incubated in pooled human serumat 37° C. as a function of time: Apparent T_(0.5)=100±4 h.

FIG. 4—A single dose of PEG-hCGL-8mut-1 can lower serum L-methionine totherapeutically relevant levels for over 15 h without dietaryintervention in a mouse model.

FIG. 5—Comparison of single doses of 200 mg/kg PEG-hCGL-NLV (opensquare) and 50 mg/kg PEG-hCGL-8mut-1 (open circle) in reducing serumL-methionine in mice on a normal diet.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention discloses compositions of matter for improvedhuman methionine-γ-lyase (hMGL) relative to the hMGL-NLV mutant (E59N,R119L, E339V). These mutants display higher catalytic activity and thuslower concentrations of therapeutic agent may be required for dosing ofpatients. These engineered human enzymes degrade the amino acidL-methionine.

These compositions provide a way to specifically target tumor cellsthrough a cancer-specific metabolic defect. While bacterial enzymes canalso perform the chemistry, their use has proven highly unstable andimmunogenic.

I. Definitions

As used herein the terms “protein” and “polypeptide” refer to compoundscomprising amino acids joined via peptide bonds and are usedinterchangeably.

As used herein, the term “fusion protein” refers to a chimeric proteincontaining proteins or protein fragments operably linked in a non-nativeway.

As used herein, the term “half-life” (½-life) refers to the time thatwould be required for the concentration of a polypeptide thereof to fallby half in vitro or in vivo, for example, after injection in a mammal.

The terms “in operable combination,” “in operable order,” and “operablylinked” refer to a linkage wherein the components so described are in arelationship permitting them to function in their intended manner, forexample, a linkage of nucleic acid sequences in such a manner that anucleic acid molecule capable of directing the transcription of a givengene and/or the synthesis of desired protein molecule, or a linkage ofamino acid sequences in such a manner so that a fusion protein isproduced.

The term “linker” is meant to refer to a compound or moiety that acts asa molecular bridge to operably link two different molecules, wherein oneportion of the linker is operably linked to a first molecule, andwherein another portion of the linker is operably linked to a secondmolecule.

The term “PEGylated” refers to conjugation with polyethylene glycol(PEG), which has been widely used as a drug carrier, given its highdegree of biocompatibility and ease of modification. PEG can be coupled(e.g., covalently linked) to active agents through the hydroxy groups atthe end of the PEG chain via chemical methods; however, PEG itself islimited to at most two active agents per molecule. In a differentapproach, copolymers of PEG and amino acids have been explored as novelbiomaterial that would retain the biocompatibility of PEG, but thatwould have the added advantage of numerous attachment points permolecule (thus providing greater drug loading), and that can besynthetically designed to suit a variety of applications.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor thereof. The polypeptide can be encoded by a full-lengthcoding sequence or by any portion of the coding sequence so as thedesired enzymatic activity is retained.

The term “native” refers to the typical form of a gene, a gene product,or a characteristic of that gene or gene product when isolated from anaturally occurring source. A native form is that which is mostfrequently observed in a natural population and is thus arbitrarilydesignated the normal or wild-type form. In contrast, the term“modified,” “variant,” or “mutant” refers to a gene or gene product thatdisplays modification in sequence and functional properties (i.e.,altered characteristics) when compared to the native gene or geneproduct.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques (see, for example, Maniatis et al., 1988 and Ausubel et al.,1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for an RNA capable of beingtranscribed. In some cases, RNA molecules are then translated into aprotein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

The term “therapeutic benefit” or “therapeutically effective” as usedthroughout this application refers to anything that promotes or enhancesthe well-being of the subject with respect to the medical treatment ofthis condition. This includes, but is not limited to, a reduction in thefrequency or severity of the signs or symptoms of a disease. Forexample, treatment of cancer may involve, for example, a reduction inthe size of a tumor, a reduction in the invasiveness of a tumor,reduction in the growth rate of the cancer, or prevention of metastasis.Treatment of cancer may also refer to prolonging survival of a subjectwith cancer.

The term “K_(M)” as used herein refers to the Michaelis-Menten constantfor an enzyme and is defined as the concentration of the specificsubstrate at which a given enzyme yields one-half its maximum velocityin an enzyme catalyzed reaction. The term “k_(cat)” as used hereinrefers to the turnover number or the number of substrate molecules eachenzyme site converts to product per unit time, and in which the enzymeis working at maximum efficiency. The term “k_(cat)/K_(M)” as usedherein is the specificity constant, which is a measure of howefficiently an enzyme converts a substrate into product.

The term “cystathionine-γ-lyase” (CGL or cystathionase) refers to anyenzyme that catalyzes the hydrolysis of cystathionine to cysteine. Forexample, it includes primate forms of cystathionine-γ-lyase, orparticularly, human forms of cystathionine-γ-lyase.

“Treatment” and “treating” refer to administration or application of atherapeutic agent to a subject or performance of a procedure or modalityon a subject for the purpose of obtaining a therapeutic benefit of adisease or health-related condition. For example, a treatment mayinclude administration of a pharmaceutically effective amount of amethioninase.

“Subject” and “patient” refer to either a human or non-human, such asprimates, mammals, and vertebrates. In particular embodiments, thesubject is a human.

II. Methionine-γ-Lyase And Cystathionine-γ-Lyase

A lyase is an enzyme that catalyzes the breaking of various chemicalbonds, often forming a new double bond or a new ring structure. Forexample, an enzyme that catalyzes this reaction would be a lyase:ATP→cAMP+PPi. Lyases differ from other enzymes in that they only requireone substrate for the reaction in one direction, but two substrates forthe reverse reaction.

A number of pyrioxal-5′-phosphate (PLP)-dependent enzymes are involvedin the metabolism of cysteine, homocysteine, and methionine, and theseenzymes form an evolutionarily related family, designated as Cys/Metmetabolism PLP-dependent enzymes. These enzymes are proteins of about400 amino acids and the PLP group is attached to a lysine residuelocated in the central location of the polypeptide. Members of thisfamily include cystathionine-γ-lyase (CGL), cystathionine-γ-synthase(CGS), cystathionine-β-lyase (CBL), methionine-γ-lyase (MGL), andO-acetylhomoserine (OAH)/O-acetyl-serine (OAS) sulfhydrylase (OSHS).Common to all of them is the formation of a Michaelis complex leading toan external substrate aldimine. The further course of the reaction isdetermined by the substrate specificity of the particular enzyme.

For example, the inventors introduced specific mutations into aPLP-dependent lyase family member, such as the humancystathionine-γ-lyase, to change its substrate specificity. In thismanner the inventors produced novel variants with the de novo ability todegrade L-Met as a substrate with substantially higher catalyticactivity than hMGL-NLV. In other embodiments, a modification of otherPLP-dependent enzymes for producing novel methionine degrading activitymay also be contemplated.

As a PLP-dependent enzyme, a methionine-γ-lyase (EC 4.4.1.11) is anenzyme that catalyzes the chemical reaction:L-methionine+H₂O→methanethiol+NH₃+2-oxobutanoate. Thus, the twosubstrates of this enzyme are L-methionine and H₂O, whereas its threeproducts are methanethiol, NH₃, and 2-oxobutanoate. This enzyme belongsto the family of lyases, specifically the class of carbon-sulfur lyases.The systematic name of this enzyme class is L-methioninemethanethiol-lyase (deaminating 2-oxobutanoate-forming). Other names incommon use include L-methioninase, methionine lyase, methioninase,methionine dethiomethylase, L-methionine-gamma-lyase, and L-methioninemethanethiol-lyase (deaminating). This enzyme participates inselenoamino acid metabolism. It employs one cofactor,pyridoxal-5′-phosphate.

Methioninase usually consists of 389-441 amino acids and forms ahomotetramer. Methioninase enzymes are generally composed of fouridentical subunits of molecular weight of ˜45 kDa (Sridhar et al., 2000;Nakamura et al., 1984). The structure of the enzyme was elucidated bycrystallization (Kudou et al., 2007). Each segment of the tetramer iscomposed of three regions: an extended N-terminal domain (residues 1-63)that includes two helices and three beta-strands, a large PLP bindingdomain (residues 64-262) that is made up of a mostly parallel sevenstranded beta-sheet that is sandwiched between eight alpha-helices, anda C-terminal domain (residues 263-398). The cofactor PLP is required forcatalytic function. Amino acids important for catalysis have beenidentified based on the structure. Tyr59 and Arg61 of neighboringsubunits, which are also strongly conserved in other c-family enzymes,contact the phosphate group of PLP. These residues are important as themain anchor within the active site. Lys240, Asp241, and Arg61 of onemonomer and Tyr114 and Cys116 of an adjacent monomer form ahydrogen-bond network in the methioninase active site that confersspecificity to the enzyme.

Cystathionine-γ-lyase (CGL or cystathionase) is an enzyme that breaksdown cystathionine into cysteine and α-ketobutyrate. Pyridoxal phosphateis a prosthetic group of this enzyme. Although mammals do not have amethioninase (MGL), they do have cystathionase with sequence,structural, and chemical homology to the bacterial MGL enzymes. As shownin the Examples, protein engineering was used to convert cystathionase,which has no activity for the degradation of L-Methionine, into anenzyme that can degrade this amino acid at a high rate.

III. Methioninase Engineering

Since humans do not produce methionine-γ-lyase (MGL or methioninase) itis necessary to engineer methioninases for human therapy that have highactivity and specificity for degrading methionine under physiologicalconditions, as well as high stability in physiological fluids, such asserum, and are also non-immunogenic because they are native proteinsthat normally elicit immunological tolerance.

Due to the undesired immunogenic effects seen in animal studies withpMGL (MGL from P. putida), it is desirable to engineer L-methioninedegradation activity in a human enzyme. Immunological tolerance to humanproteins makes it likely that such an enzyme will be non-immunogenic orminimally immunogenic and therefore well tolerated.

Certain aspects of novel enzymes with MGL activity as engineeredmethioninase address these needs. Although mammals do not have a MGL,they do have a cystathionine-γ-lyase (CGL) that has sequence,structural, and chemical homology to the bacterial MGL enzymes. CGL is atetramer that catalyzes the last step in the mammalian transsulfurationpathway (Rao et al., 1990). CGL catalyzes the conversion ofL-cystathionine to L-cysteine, alpha-ketobutyrate, and ammonia. Thehuman CGL (hCGL) cDNA had previously been cloned and expressed, but withrelatively low yields (˜5 mg/L culture) (Lu et al., 1992; Steegborn etal., 1999).

For example, there have been provided methods and compositions relatedto a primate (particularly human) cystathionine-γ-lyase (CGL orcystathionase) modified via mutagenesis to hydrolyze methionine withhigh efficiency, while the cystathionine-γ-lyase does not exhibitmethioninase activity in its native form.

Some embodiments concern modified proteins and polypeptides. Particularembodiments concern a modified protein or polypeptide that exhibits atleast one functional activity that is comparable to the unmodifiedversion, preferably, the methioninase enzyme activity. In furtheraspects, the protein or polypeptide may be further modified to increaseserum stability. Thus, when the present application refers to thefunction or activity of “modified protein” or a “modified polypeptide,”one of ordinary skill in the art would understand that this includes,for example, a protein or polypeptide that possesses an additionaladvantage over the unmodified protein or polypeptide, such as themethioninase enzyme activity. In certain embodiments, the unmodifiedprotein or polypeptide is a native cystathionine-γ-lyase, specifically ahuman cystathionine-γ-lyase. It is specifically contemplated thatembodiments concerning a “modified protein” may be implemented withrespect to a “modified polypeptide,” and vice versa.

Determination of activity may be achieved using assays familiar to thoseof skill in the art, particularly with respect to the protein'sactivity, and may include for comparison purposes, for example, the useof native and/or recombinant versions of either the modified orunmodified protein or polypeptide. For example, the methioninaseactivity may be determined by any assay to detect the production of anysubstrates resulting from conversion of methionine, such asalpha-ketobutyrate, methanethiol, and/or ammonia.

In certain embodiments, a modified polypeptide, such as a modifiedcystathionine-γ-lyase, may be identified based on its increase inmethionine degrading activity. For example, substrate recognition sitesof the unmodified polypeptide may be identified. This identification maybe based on structural analysis or homology analysis. A population ofmutants involving modifications of such substrate recognitions sites maybe generated. In a further embodiment, mutants with increased methioninedegrading activity may be selected from the mutant population. Selectionof desired mutants may include methods, such as detection of byproductsor products from methionine degradation.

Modified proteins may possess deletions and/or substitutions of aminoacids; thus, a protein with a deletion, a protein with a substitution,and a protein with a deletion and a substitution are modified proteins.In some embodiments, these modified proteins may further includeinsertions or added amino acids, such as with fusion proteins orproteins with linkers, for example. A “modified deleted protein” lacksone or more residues of the native protein, but may possess thespecificity and/or activity of the native protein. A “modified deletedprotein” may also have reduced immunogenicity or antigenicity. Anexample of a modified deleted protein is one that has an amino acidresidue deleted from at least one antigenic region, that is, a region ofthe protein determined to be antigenic in a particular organism, such asthe type of organism that may be administered the modified protein.

Substitution or replacement variants typically contain the exchange ofone amino acid for another at one or more sites within the protein andmay be designed to modulate one or more properties of the polypeptide,particularly its effector functions and/or bioavailability.Substitutions may or may not be conservative, that is, one amino acid isreplaced with one of similar shape and charge. Conservativesubstitutions are well known in the art and include, for example, thechanges of: alanine to serine; arginine to lysine; asparagine toglutamine or histidine; aspartate to glutamate; cysteine to serine;glutamine to asparagine; glutamate to aspartate; glycine to proline;histidine to asparagine or glutamine; isoleucine to leucine or valine;leucine to valine or isoleucine; lysine to arginine; methionine toleucine or isoleucine; phenylalanine to tyrosine, leucine, ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

In addition to a deletion or substitution, a modified protein maypossess an insertion of residues, which typically involves the additionof at least one residue in the polypeptide. This may include theinsertion of a targeting peptide or polypeptide or simply a singleresidue. Terminal additions, called fusion proteins, are discussedbelow.

The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein. Accordingly, sequences thathave between about 70% and about 80%, or between about 81% and about90%, or even between about 91% and about 99% of amino acids that areidentical or functionally equivalent to the amino acids of a controlpolypeptide are included, provided the biological activity of theprotein is maintained. A modified protein may be biologicallyfunctionally equivalent to its native counterpart in certain aspects.

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

IV. Enzymatic L-Methionine Depletion For Therapy

In certain aspects, the polypeptides may be used for the treatment ofdiseases, including cancers that are sensitive to methionine depletion,such as hepatocellular carcinoma, melanoma, and renal cell carcinoma,with novel enzymes that deplete L-methionine. The invention specificallydiscloses treatment methods using modified cystathionine-γ-lyase withmethionine degrading activity. As described below, as currentlyavailable methionine-γ-lyases are typically bacterially-derivedproteins, there remain several problems for their use in human therapy.Certain embodiments of the present invention provide novel enzymes withmethionine-γ-lyase activity for increased therapeutic efficacy.

Methionine (L-Met) depletion has long been studied as a potentialtreatment for cancer. While L-Met is an essential amino acid, manymalignant human cell lines and tumors have been shown to have arelatively greater requirement for methionine (Halpern et al., 1974;Kreis and Goodenow, 1978; Breillout et al., 1990; Kreis et al., 1980;Kreis, 1979). Methionine-dependent tumor cell lines present no or lowlevels of methionine synthase, the enzyme that normally recycleshomocysteine back to L-Met (Halpern et al., 1974; Ashe et al., 1974).Most normal cells can grow on precursors, such as homocysteine andhomocystine, whereas many malignant cells must scavenge L-Met directlyfrom their extracellular environment. Also, any rapidly growing neoplasmcan be adversely affected by the lack of essential building blocksnecessary for growth. Methionine is particularly important as itsdepletion leads not only to diminished protein synthesis, but alsodysregulated S-adenosylmethionine (SAM)-dependent methylation pathways,which are particularly important for gene regulation.

The differences in methionine requirements between normal and cancercells provide a therapeutic opportunity. Enzymatic methionine depletionhas been explored in a number of animal model studies as well as Phase Iclinical trials (Tan et al., 1997a; Tan et al., 1996a; Lishko et al.,1993; Tan et al., 1996b; Yoshioka et al., 1998; Yang et al., 2004a; Yanget al., 2004b; Tan et al., 1997b).

Because humans lack a methionine hydrolyzing enzyme, bacterialL-methionine-γ-lyases, MGL, from various sources have been evaluated forcancer therapy. Methionine-γ-lyase catalyzes the conversion ofmethionine to methanethiol, alpha-ketobutyrate, and ammonia. Bacterialenzymes from various sources have been purified and tested as methioninedepleting agents against cancer cell lines. The P. putida (pMGL) sourcewas selected for therapeutic applications due to its high catalyticactivity, low K_(M) value, and relatively high k_(cat) value (Esaki andSoda, 1987; Ito et al., 1976), in comparison to other sources.Furthermore, the gene for pMGL has been cloned into E. coli and theprotein was expressed at a high protein yield (Tan et al., 1997a; Horiet al., 1996).

In vivo studies have been performed on animal models, as well as humans.Tan et al. (1997a) performed studies with human tumors xenografted intonude mice and found that lung, colon, kidney, brain, prostate, andmelanoma cancers were all sensitive to pMGL. Additionally, no toxicitywas detected at effective doses, as was determined by an absence ofweight loss in the animals. Half-life in these experiments wasdetermined to be only 2 h as measured from collected blood samples.Additionally, infusion of PLP is required in order to maintain MGLactivity. In spite of the very short half-life, Tan et al. (1997a)reported inhibition of tumor growth in comparison to a saline control.

Yang et al. (2004b) studied the pharmacokinetics, the pharmacodynamicsin terms of methionine depletion, the antigenicity, and toxicity of MGLin a primate model. Dose-ranging studies were performed at 1000-4000units/kg administered intravenously. The highest dose was able to reduceplasma methionine to an undetectable level (less than 0.5 μM) by 30 minafter injection, with the methionine level remaining undetectable for 8h. Pharmacokinetic analysis showed that pMGL was eliminated with ahalf-life of 2.5 h. An administration of that dose every 8 h/day for 2weeks resulted in a steady-state depletion of plasma methionine to lessthan 2 μM. Mild toxicity was observed through decreased food intake andslight weight loss. Unfortunately, re-challenge on day 28 resulted inanaphylactic shock and death in one animal indicating that pMGL ishighly immunogenic, which is a significant disadvantage for humantherapy. Subsequent pretreatment with hydrocortisone prevented theanaphylactic reaction, although vomiting was frequently observed.Additional re-challenges were carried out at days 66, 86, and 116.Anti-rMGL antibodies were detected after the first challenge, andincreased in concentration for the duration of treatment.

In response to these observed obstacles to therapeutic implementation ofMGL, Yang et al. (2004b) studied the PEGylation of the enzyme and itseffect on half-life and immunogenicity. The enzyme was coupled tomethoxypolyethylene glycol succinimidyl glutarate (MEGC-PEG-5000). Doseranging studies were again performed and 4000 units/kg (90 mg/kg) wassufficient to reduce plasma methionine to <5 μmol/L for 12 h.Pharmacokinetic analysis showed a 36-fold improvement in the serumclearance half-life of the PEGylated enzyme, as compared to theunPEGylated enzyme. PEGylating also attenuated immunogenicity somewhatas only slight toxicities of decreased food intake and minor weight losswere observed. However, the activity half-life was not improved as L-Metlevels were only kept below detection levels for 12 h as opposed to 8 hfor the unPEGylated enzyme. These results, though promising for the useof an L-Met depleting enzyme as an anti-neoplastic agent, are challengedby significant shortcomings of immunogenicity and pharmacokinetics.

Certain aspects of the present invention provide a modifiedcystathionine-γ-lyase with methionine degrading activity for treatingdiseases, such as tumors. Particularly, the modified polypeptide mayhave human polypeptide sequences and thus may prevent allergic reactionsin human patients, allow repeated dosing, and increase the therapeuticefficacy.

Tumors for which the present treatment methods are useful include anymalignant cell type, such as those found in a solid tumor or ahematological tumor. Exemplary solid tumors can include, but are notlimited to, a tumor of an organ selected from the group consisting ofpancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney,larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.Exemplary hematological tumors include tumors of the bone marrow, T or Bcell malignancies, leukemias, lymphomas, blastomas, myelomas, and thelike. Further examples of cancers that may be treated using the methodsprovided herein include, but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, leukemia, squamous cell cancer, lung cancer(including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer and gastrointestinal stromal cancer),pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, varioustypes of head and neck cancer, melanoma, superficial spreading melanoma,lentigo malignant melanoma, acral lentiginous melanomas, nodularmelanomas, as well as B-cell lymphoma (including low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's macroglobulinemia), chroniclymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairycell leukemia, multiple myeloma, acute myeloid leukemia (AML) andchronic myeloblastic leukemia.

The cancer may specifically be of the following histological type,though it is not limited to these: neoplasm, malignant; carcinoma;carcinoma, undifferentiated; giant and spindle cell carcinoma; smallcell carcinoma; papillary carcinoma; squamous cell carcinoma;lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma;transitional cell carcinoma; papillary transitional cell carcinoma;adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant;thecoma, malignant; granulosa cell tumor, malignant; androblastoma,malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipidcell tumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; malignantmelanoma in giant pigmented nevus; epithelioid cell melanoma; bluenevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumor, malignant; mullerian mixed tumor; nephroblastoma;hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor,malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma,malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant;hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma;hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignantlymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse;malignant lymphoma, follicular; mycosis fungoides; other specifiednon-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mastcell sarcoma; immunoproliferative small intestinal disease; leukemia;lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcomacell leukemia; myeloid leukemia; basophilic leukemia; eosinophilicleukemia; monocytic leukemia; mast cell leukemia; megakaryoblasticleukemia; myeloid sarcoma; and hairy cell leukemia.

The engineered human methioninase derived from cystathionase may be usedherein as an antitumor agent in a variety of modalities for depletingmethionine from a tumor cell, tumor tissue, or the circulation of amammal with cancer, or for depletion of methionine where its depletionis considered desirable.

Depletion can be conducted in vivo in the circulation of a mammal, invitro in cases where methionine depletion in tissue culture or otherbiological mediums is desired, and in ex vivo procedures wherebiological fluids, cells, or tissues are manipulated outside the bodyand subsequently returned to the body of the patient mammal. Depletionof methionine from circulation, culture media, biological fluids, orcells is conducted to reduce the amount of methionine accessible to thematerial being treated, and therefore comprises contacting the materialto be depleted with a methionine-depleting amount of the engineeredhuman methioninase under methionine-depleting conditions as to degradethe ambient methionine in the material being contacted.

Because tumor cells are dependent upon their nutrient medium formethionine, the depletion may be directed to the nutrient source for thecells, and not necessarily the cells themselves. Therefore, in an invivo application, treating a tumor cell includes contacting the nutrientmedium for a population of tumor cells with the engineered methioninase.In this embodiment, the medium may be blood, lymphatic fluid, spinalfluid and the like bodily fluid where methionine depletion is desired.

A methionine-depleting efficiency can vary widely depending upon theapplication, and typically depends upon the amount of methionine presentin the material, the desired rate of depletion, and the tolerance of thematerial for exposure to methioninase. Methionine levels in a material,and therefore rates of methionine depletion from the material, canreadily be monitored by a variety of chemical and biochemical methodswell known in the art. Exemplary methionine-depleting amounts aredescribed further herein, and can range from 0.001 to 100 units (U) ofengineered methioninase, preferably about 0.01 to 10 U, and morepreferably about 0.1 to 5 U engineered methioninase per milliliter (mL)of material to be treated.

Methionine-depleting conditions are buffer and temperature conditionscompatible with the biological activity of a methioninase enzyme, andinclude moderate temperature, salt, and pH conditions compatible withthe enzyme, for example, physiological conditions. Exemplary conditionsinclude about 4-40° C., ionic strength equivalent to about 0.05 to 0.2 MNaCl, and a pH of about 5 to 9, while physiological conditions areincluded.

In a particular embodiment, the invention contemplates methods of usingengineered methioninase as an antitumor agent, and therefore comprisescontacting a population of tumor cells with a therapeutically effectiveamount of engineered methioninase for a time period sufficient toinhibit tumor cell growth.

In one embodiment, the contacting in vivo is accomplished byadministering, by intravenous or intraperitoneal injection, atherapeutically effective amount of a physiologically tolerablecomposition comprising an engineered methioninase of this invention to apatient, thereby depleting the circulating methionine source of thetumor cells present in the patient. The contacting of engineeredmethioninase can also be accomplished by administering the engineeredmethioninase into the tissue containing the tumor cells.

A therapeutically effective amount of an engineered methioninase is apredetermined amount calculated to achieve the desired effect, i.e., todeplete methionine in the tumor tissue or in a patient's circulation,and thereby cause the tumor cells to stop dividing. Thus, the dosageranges for the administration of engineered methioninase of theinvention are those large enough to produce the desired effect in whichthe symptoms of tumor cell division and cell cycling are reduced. Thedosage should not be so large as to cause adverse side effects, such ashyperviscosity syndromes, pulmonary edema, congestive heart failure, andthe like. Generally, the dosage will vary with age of, condition of, sexof, and extent of the disease in the patient and can be determined byone of skill in the art. The dosage can be adjusted by the individualphysician in the event of any complication.

For example, a therapeutically effective amount of an engineeredmethioninase may be an amount such that when administered in aphysiologically tolerable composition is sufficient to achieve anintravascular (plasma) or local concentration of from about 0.001 toabout 100 units (U) per mL, preferably above about 0.1 U, and morepreferably above 1 U engineered methioninase per mL. Typical dosages canbe administered based on body weight, and are in the range of about5-1000 U/kilogram (kg)/day, preferably about 5-100 U/kg/day, morepreferably about 10-50 U/kg/day, and more preferably about 20-40U/kg/day.

The engineered methioninase can be administered parenterally byinjection or by gradual infusion over time. The engineered methioninasecan be administered intravenously, intraperitoneally, orally,intramuscularly, subcutaneously, intracavity, transdermally, dermally,can be delivered by peristaltic means, can be injected directly into thetissue containing the tumor cells, or can be administered by a pumpconnected to a catheter that may contain a potential biosensor ormethionine.

The therapeutic compositions containing engineered methioninase areconventionally administered intravenously, as by injection of a unitdose, for example. The term “unit dose” when used in reference to atherapeutic composition refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent, i.e.,carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for initial administration and boostershots are also contemplated and are typified by an initialadministration followed by repeated doses at one or more hour intervalsby a subsequent injection or other administration. Exemplary multipleadministrations are described herein and are particularly preferred tomaintain continuously high serum and tissue levels of engineeredmethioninase and conversely low serum and tissue levels of methionine.Alternatively, continuous intravenous infusion sufficient to maintainconcentrations in the blood in the ranges specified for in vivotherapies are contemplated.

V. Conjugates

Compositions and methods of the present invention involve furthermodification of the engineered methioninase for improvement, such as byforming conjugates with heterologous peptide segments or polymers, suchas polyethylene glycol. In further aspects, the engineered methioninasemay be linked to PEG to increase the hydrodynamic radius of the enzymeand hence increase the serum persistence. In certain aspects, thedisclosed polypeptide may be conjugated to any targeting agent, such asa ligand having the ability to specifically and stably bind to anexternal receptor or binding site on a tumor cell (U.S. Patent Publ.2009/0304666).

A. Fusion Proteins

Certain embodiments of the present invention concern fusion proteins.These molecules may have the engineered human methioninase linked at theN- or C-terminus to a heterologous domain. For example, fusions may alsoemploy leader sequences from other species to permit the recombinantexpression of a protein in a heterologous host. Another useful fusionincludes the addition of a protein affinity tag, such as a serum albuminaffinity tag or six histidine residues, or an immunologically activedomain, such as an antibody epitope, preferably cleavable, to facilitatepurification of the fusion protein. Non-limiting affinity tags includepolyhistidine, chitin binding protein (CBP), maltose binding protein(MBP), and glutathione-S-transferase (GST).

In a particular embodiment, the engineered human methioninase may belinked to a peptide that increases the in vivo half-life, such as anXTEN polypeptide (Schellenberger et al., 2009), IgG Fc domain, albumin,or an albumin binding peptide.

Methods of generating fusion proteins are well known to those of skillin the art. Such proteins can be produced, for example, by de novosynthesis of the complete fusion protein, or by attachment of the DNAsequence encoding the heterologous domain, followed by expression of theintact fusion protein.

Production of fusion proteins that recover the functional activities ofthe parent proteins may be facilitated by connecting genes with abridging DNA segment encoding a peptide linker that is spliced betweenthe polypeptides connected in tandem. The linker would be of sufficientlength to allow proper folding of the resulting fusion protein.

B. Linkers

In certain embodiments, the engineered methioninase may be chemicallyconjugated using bifunctional cross-linking reagents or fused at theprotein level with peptide linkers.

Bifunctional cross-linking reagents have been extensively used for avariety of purposes, including preparation of affinity matrices,modification and stabilization of diverse structures, identification ofligand and receptor binding sites, and structural studies. Suitablepeptide linkers may also be used to link the engineered methioninase,such as Gly-Ser linkers.

Homobifunctional reagents that carry two identical functional groupsproved to be highly efficient in inducing cross-linking betweenidentical and different macromolecules or subunits of a macromolecule,and linking of polypeptide ligands to their specific binding sites.Heterobifunctional reagents contain two different functional groups. Bytaking advantage of the differential reactivities of the two differentfunctional groups, cross-linking can be controlled both selectively andsequentially. The bifunctional cross-linking reagents can be dividedaccording to the specificity of their functional groups, e.g., amino-,sulfhydryl-, guanidine-, indole-, carboxyl-specific groups. Of these,reagents directed to free amino groups have become especially popularbecause of their commercial availability, ease of synthesis, and themild reaction conditions under which they can be applied.

A majority of heterobifunctional cross-linking reagents contain aprimary amine-reactive group and a thiol-reactive group. In anotherexample, heterobifunctional cross-linking reagents and methods of usingthe cross-linking reagents are described (U.S. Pat. No. 5,889,155,specifically incorporated herein by reference in its entirety). Thecross-linking reagents combine a nucleophilic hydrazide residue with anelectrophilic maleimide residue, allowing coupling, in one example, ofaldehydes to free thiols. The cross-linking reagent can be modified tocross-link various functional groups.

Additionally, any other linking/coupling agents and/or mechanisms knownto those of skill in the art may be used to combine human engineeredmethioninase, such as, for example, antibody-antigen interaction, avidinbiotin linkages, amide linkages, ester linkages, thioester linkages,ether linkages, thioether linkages, phosphoester linkages, phosphoramidelinkages, anhydride linkages, disulfide linkages, ionic and hydrophobicinteractions, bispecific antibodies and antibody fragments, orcombinations thereof.

It is preferred that a cross-linker having reasonable stability in bloodwill be employed. Numerous types of disulfide-bond containing linkersare known that can be successfully employed to conjugate targeting andtherapeutic/preventative agents. Linkers that contain a disulfide bondthat is sterically hindered may prove to give greater stability in vivo.These linkers are thus one group of linking agents.

In addition to hindered cross-linkers, non-hindered linkers also can beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP, and 2-iminothiolane (Wawrzynczak and Thorpe, 1987). The use ofsuch cross-linkers is well understood in the art. Another embodimentinvolves the use of flexible linkers.

Once chemically conjugated, the peptide generally will be purified toseparate the conjugate from unconjugated agents and from othercontaminants. A large number of purification techniques are availablefor use in providing conjugates of a sufficient degree of purity torender them clinically useful.

Purification methods based upon size separation, such as gel filtration,gel permeation, or high performance liquid chromatography, willgenerally be of most use. Other chromatographic techniques, such asBlue-Sepharose separation, may also be used. Conventional methods topurify the fusion proteins from inclusion bodies may be useful, such asusing weak detergents, such as sodium N-lauroyl-sarcosine (SLS).

C. PEGylation

In certain aspects of the invention, methods and compositions related toPEGylation of engineered methioninase are disclosed. For example, theengineered methioninase may be PEGylated in accordance with the methodsdisclosed herein.

PEGylation is the process of covalent attachment of poly(ethyleneglycol) polymer chains to another molecule, normally a drug ortherapeutic protein. PEGylation is routinely achieved by incubation of areactive derivative of PEG with the target macromolecule. The covalentattachment of PEG to a drug or therapeutic protein can “mask” the agentfrom the host's immune system (reduced immunogenicity and antigenicity)or increase the hydrodynamic size (size in solution) of the agent, whichprolongs its circulatory time by reducing renal clearance. PEGylationcan also provide water solubility to hydrophobic drugs and proteins.

The first step of the PEGylation is the suitable functionalization ofthe PEG polymer at one or both terminals. PEGs that are activated ateach terminus with the same reactive moiety are known as“homobifunctional,” whereas if the functional groups present aredifferent, then the PEG derivative is referred as “heterobifunctional”or “heterofunctional.” The chemically active or activated derivatives ofthe PEG polymer are prepared to attach the PEG to the desired molecule.

The choice of the suitable functional group for the PEG derivative isbased on the type of available reactive group on the molecule that willbe coupled to the PEG. For proteins, typical reactive amino acidsinclude lysine, cysteine, histidine, arginine, aspartic acid, glutamicacid, serine, threonine, and tyrosine. The N-terminal amino group andthe C-terminal carboxylic acid can also be used.

The techniques used to form first generation PEG derivatives aregenerally reacting the PEG polymer with a group that is reactive withhydroxyl groups, typically anhydrides, acid chlorides, chloroformates,and carbonates. In the second generation PEGylation chemistry moreefficient functional groups, such as aldehyde, esters, amides, etc., aremade available for conjugation.

As applications of PEGylation have become more and more advanced andsophisticated, there has been an increase in need for heterobifunctionalPEGs for conjugation. These heterobifunctional PEGs are very useful inlinking two entities, where a hydrophilic, flexible, and biocompatiblespacer is needed. Preferred end groups for heterobifunctional PEGs aremaleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acids,and NHS esters.

The most common modification agents, or linkers, are based on methoxyPEG (mPEG) molecules. Their activity depends on adding aprotein-modifying group to the alcohol end. In some instancespolyethylene glycol (PEG diol) is used as the precursor molecule. Thediol is subsequently modified at both ends in order to make a hetero- orhomo-dimeric PEG-linked molecule.

Proteins are generally PEGylated at nucleophilic sites, such asunprotonated thiols (cysteinyl residues) or amino groups. Examples ofcysteinyl-specific modification reagents include PEG maleimide, PEGiodoacetate, PEG thiols, and PEG vinylsulfone. All four are stronglycysteinyl-specific under mild conditions and neutral to slightlyalkaline pH but each has some drawbacks. The thioether formed with themaleimides can be somewhat unstable under alkaline conditions so theremay be some limitation to formulation options with this linker. Thecarbamothioate linkage formed with iodo PEGs is more stable, but freeiodine can modify tyrosine residues under some conditions. PEG thiolsform disulfide bonds with protein thiols, but this linkage can also beunstable under alkaline conditions. PEG-vinylsulfone reactivity isrelatively slow compared to maleimide and iodo PEG; however, thethioether linkage formed is quite stable. Its slower reaction rate alsocan make the PEG-vinylsulfone reaction easier to control.

Site-specific PEGylation at native cysteinyl residues is seldom carriedout, since these residues are usually in the form of disulfide bonds orare required for biological activity. On the other hand, site-directedmutagenesis can be used to incorporate cysteinyl PEGylation sites forthiol-specific linkers. The cysteine mutation must be designed such thatit is accessible to the PEGylation reagent and is still biologicallyactive after PEGylation.

Amine-specific modification agents include PEG NHS ester, PEG tresylate,PEG aldehyde, PEG isothiocyanate, and several others. All react undermild conditions and are very specific for amino groups. The PEG NHSester is probably one of the more reactive agents; however, its highreactivity can make the PEGylation reaction difficult to control on alarge scale. PEG aldehyde forms an imine with the amino group, which isthen reduced to a secondary amine with sodium cyanoborohydride. Unlikesodium borohydride, sodium cyanoborohydride will not reduce disulfidebonds. However, this chemical is highly toxic and must be handledcautiously, particularly at lower pH where it becomes volatile.

Due to the multiple lysine residues on most proteins, site-specificPEGylation can be a challenge. Fortunately, because these reagents reactwith unprotonated amino groups, it is possible to direct the PEGylationto lower-pK amino groups by performing the reaction at a lower pH.Generally the pK of the alpha-amino group is 1-2 pH units lower than theepsilon-amino group of lysine residues. By PEGylating the molecule at pH7 or below, high selectivity for the N-terminus frequently can beattained. However, this is only feasible if the N-terminal portion ofthe protein is not required for biological activity. Still, thepharmacokinetic benefits from PEGylation frequently outweigh asignificant loss of in vitro bioactivity, resulting in a product withmuch greater in vivo bioactivity regardless of PEGylation chemistry.

There are several parameters to consider when developing a PEGylationprocedure. Fortunately, there are usually no more than four or five keyparameters. The “design of experiments” approach to optimization ofPEGylation conditions can be very useful. For thiol-specific PEGylationreactions, parameters to consider include: protein concentration,PEG-to-protein ratio (on a molar basis), temperature, pH, reaction time,and in some instances, the exclusion of oxygen. (Oxygen can contributeto intermolecular disulfide formation by the protein, which will reducethe yield of the PEGylated product.) The same factors should beconsidered (with the exception of oxygen) for amine-specificmodification except that pH may be even more critical, particularly whentargeting the N-terminal amino group.

For both amine- and thiol-specific modifications, the reactionconditions may affect the stability of the protein. This may limit thetemperature, protein concentration, and pH. In addition, the reactivityof the PEG linker should be known before starting the PEGylationreaction. For example, if the PEGylation agent is only 70% active, theamount of PEG used should ensure that only active PEG molecules arecounted in the protein-to-PEG reaction stoichiometry.

VI. Proteins And Peptides

In certain embodiments, the present invention concerns novelcompositions comprising at least one protein or peptide, such as anengineered methioninase. These peptides may be comprised in a fusionprotein or conjugated to an agent as described supra.

As used herein, a protein or peptide generally refers, but is notlimited to, a protein of greater than about 200 amino acids, up to afull-length sequence translated from a gene; a polypeptide of greaterthan about 100 amino acids; and/or a peptide of from about 3 to about100 amino acids. For convenience, the terms “protein,” “polypeptide,”and “peptide” are used interchangeably herein.

As used herein, an “amino acid residue” refers to any naturallyoccurring amino acid, any amino acid derivative, or any amino acid mimicknown in the art. In certain embodiments, the residues of the protein orpeptide are sequential, without any non-amino acids interrupting thesequence of amino acid residues. In other embodiments, the sequence maycomprise one or more non-amino acid moieties. In particular embodiments,the sequence of residues of the protein or peptide may be interrupted byone or more non-amino acid moieties.

Accordingly, the term “protein or peptide” encompasses amino acidsequences comprising at least one of the 20 common amino acids found innaturally occurring proteins, or at least one modified or unusual aminoacid.

Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides, orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, or the chemical synthesisof proteins or peptides. The nucleotide and protein, polypeptide, andpeptide sequences corresponding to various genes have been previouslydisclosed, and may be found at computerized databases known to those ofordinary skill in the art. One such database is the National Center forBiotechnology Information's Genbank and GenPept databases (available onthe world wide web at ncbi.nlm.nih.gov/). The coding regions for knowngenes may be amplified and/or expressed using the techniques disclosedherein or as would be known to those of ordinary skill in the art.Alternatively, various commercial preparations of proteins,polypeptides, and peptides are known to those of skill in the art.

VII. Nucleic Acids And Vectors

In certain aspects of the invention, nucleic acid sequences encoding anengineered methioninase or a fusion protein containing an engineeredhuman methioninase may be disclosed. Depending on which expressionsystem is used, nucleic acid sequences can be selected based onconventional methods. For example, if the engineered methioninase isderived from human cystathionase and contains multiple codons that arerarely utilized in E. coli, then that may interfere with expression.Therefore, the respective genes or variants thereof may be codonoptimized for E. coli expression. Various vectors may be also used toexpress the protein of interest, such as engineered methioninase.Exemplary vectors include, but are not limited, plasmid vectors, viralvectors, transposon, or liposome-based vectors.

VIII. Host Cells

Host cells may be any that may be transformed to allow the expressionand secretion of engineered methioninase and conjugates thereof. Thehost cells may be bacteria, mammalian cells, yeast, or filamentousfungi. Various bacteria include Escherichia and Bacillus. Yeastsbelonging to the genera Saccharomyces, Kiuyveromyces, Hansenula, orPichia would find use as an appropriate host cell. Various species offilamentous fungi may be used as expression hosts, including thefollowing genera: Aspergillus, Trichoderma, Neurospora, Penicillium,Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, andPyricularia.

Examples of usable host organisms include bacteria, e.g., Escherichiacoli MC1061, derivatives of Bacillus subtilis BRB1 (Sibakov et al.,1984), Staphylococcus aureus SAI123 (Lordanescu, 1975) or Streptococcuslividans (Hopwood et al., 1985); yeasts, e.g., Saccharomyces cerevisiaeAH 22 (Mellor et al., 1983) or Schizosaccharomyces pombe; andfilamentous fungi, e.g., Aspergillus nidulans, Aspergillus awamori(Ward, 1989), or Trichoderma reesei (Penttila et al., 1987; Harkki etal., 1989).

Examples of mammalian host cells include Chinese hamster ovary cells(CHO-K1; ATCC CCL61), rat pituitary cells (GH1; ATCC CCL82), HeLa S3cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCCCRL 1548),SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650), and murineembryonic cells (NIH-3T3; ATCC CRL 1658). The foregoing is meant to beillustrative but not limitative of the many possible host organismsknown in the art. In principle, all hosts capable of secretion can beused whether prokaryotic or eukaryotic.

Mammalian host cells expressing the engineered methioninases and/ortheir fusion proteins are cultured under conditions typically employedto culture the parental cell line. Generally, cells are cultured in astandard medium containing physiological salts and nutrients, such asstandard RPMI, MEM, IMEM, or DMEM, typically supplemented with 5%-10%serum, such as fetal bovine serum. Culture conditions are also standard,e.g., cultures are incubated at 37° C. in stationary or roller culturesuntil desired levels of the proteins are achieved.

IX. Protein Purification

Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the homogenization andcrude fractionation of the cells, tissue, or organ to polypeptide andnon-polypeptide fractions. The protein or polypeptide of interest may befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity) unless otherwise specified. Analytical methods particularlysuited to the preparation of a pure peptide are ion-exchangechromatography, gel exclusion chromatography, polyacrylamide gelelectrophoresis, affinity chromatography, immunoaffinity chromatography,and isoelectric focusing. A particularly efficient method of purifyingpeptides is fast-performance liquid chromatography (FPLC) or evenhigh-performance liquid chromatography (HPLC).

A purified protein or peptide is intended to refer to a composition,isolatable from other components, wherein the protein or peptide ispurified to any degree relative to its naturally-obtainable state. Anisolated or purified protein or peptide, therefore, also refers to aprotein or peptide free from the environment in which it may naturallyoccur. Generally, “purified” will refer to a protein or peptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a composition in which theprotein or peptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, or more of the proteins in the composition.

Various techniques suitable for use in protein purification are wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulfate, PEG, antibodies and the like, or byheat denaturation, followed by centrifugation; chromatography steps,such as ion exchange, gel filtration, reverse phase, hydroxyapatite, andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of these and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

Various methods for quantifying the degree of purification of theprotein or peptide are known to those of skill in the art in light ofthe present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity therein,assessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification, andwhether or not the expressed protein or peptide exhibits a detectableactivity.

There is no general requirement that the protein or peptide will alwaysbe provided in its most purified state. Indeed, it is contemplated thatless substantially purified products may have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

In certain embodiments a protein or peptide may be isolated or purified,for example, an engineered methioninase, a fusion protein containing theengineered methioninase, or an engineered methioninase post PEGylation.For example, a His tag or an affinity epitope may be comprised in suchan engineered methioninase to facilitate purification. Affinitychromatography is a chromatographic procedure that relies on thespecific affinity between a substance to be isolated and a molecule towhich it can specifically bind. This is a receptor-ligand type ofinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (e.g., altered pH, ionic strength, temperature, etc.). Thematrix should be a substance that does not adsorb molecules to anysignificant extent and that has a broad range of chemical, physical, andthermal stability. The ligand should be coupled in such a way as to notaffect its binding properties. The ligand should also provide relativelytight binding. It should be possible to elute the substance withoutdestroying the sample or the ligand.

Size exclusion chromatography (SEC) is a chromatographic method in whichmolecules in solution are separated based on their size, or in moretechnical terms, their hydrodynamic volume. It is usually applied tolarge molecules or macromolecular complexes, such as proteins andindustrial polymers. Typically, when an aqueous solution is used totransport the sample through the column, the technique is known as gelfiltration chromatography, versus the name gel permeationchromatography, which is used when an organic solvent is used as amobile phase.

The underlying principle of SEC is that particles of different sizeswill elute (filter) through a stationary phase at different rates. Thisresults in the separation of a solution of particles based on size.Provided that all the particles are loaded simultaneously or nearsimultaneously, particles of the same size should elute together. Eachsize exclusion column has a range of molecular weights that can beseparated. The exclusion limit defines the molecular weight at the upperend of this range and is where molecules are too large to be trapped inthe stationary phase. The permeation limit defines the molecular weightat the lower end of the range of separation and is where molecules of asmall enough size can penetrate into the pores of the stationary phasecompletely and all molecules below this molecular mass are so small thatthey elute as a single band.

High-performance liquid chromatography (or high-pressure liquidchromatography, HPLC) is a form of column chromatography used frequentlyin biochemistry and analytical chemistry to separate, identify, andquantify compounds. HPLC utilizes a column that holds chromatographicpacking material (stationary phase), a pump that moves the mobilephase(s) through the column, and a detector that shows the retentiontimes of the molecules. Retention time varies depending on theinteractions between the stationary phase, the molecules being analyzed,and the solvent(s) used.

X. Pharmaceutical Compositions

It is contemplated that the novel methioninase can be administeredsystemically or locally to inhibit tumor cell growth and, mostpreferably, to kill cancer cells in cancer patients with locallyadvanced or metastatic cancers. They can be administered intravenously,intrathecally, and/or intraperitoneally. They can be administered aloneor in combination with anti-proliferative drugs. In one embodiment, theyare administered to reduce the cancer load in the patient prior tosurgery or other procedures. Alternatively, they can be administeredafter surgery to ensure that any remaining cancer (e.g., cancer that thesurgery failed to eliminate) does not survive.

It is not intended that the present invention be limited by theparticular nature of the therapeutic preparation. For example, suchcompositions can be provided in formulations together withphysiologically tolerable liquid, gel, or solid carriers, diluents, andexcipients. These therapeutic preparations can be administered tomammals for veterinary use, such as with domestic animals, and clinicaluse in humans in a manner similar to other therapeutic agents. Ingeneral, the dosage required for therapeutic efficacy will varyaccording to the type of use and mode of administration, as well as theparticularized requirements of individual subjects.

Such compositions are typically prepared as liquid solutions orsuspensions, for use as injectables. Suitable diluents and excipientsare, for example, water, saline, dextrose, glycerol, or the like, andcombinations thereof. In addition, if desired, the compositions maycontain minor amounts of auxiliary substances, such as wetting oremulsifying agents, stabilizing agents, or pH buffering agents.

Where clinical applications are contemplated, it may be necessary toprepare pharmaceutical compositions comprising proteins, antibodies, anddrugs in a form appropriate for the intended application. Generally,pharmaceutical compositions may comprise an effective amount of one ormore engineered methioninase or additional agents dissolved or dispersedin a pharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic, or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcontains at least one engineered methioninase isolated by the methoddisclosed herein, or additional active ingredient will be known to thoseof skill in the art in light of the present disclosure, as exemplifiedby Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporatedherein by reference. Moreover, for animal (e.g., human) administration,it will be understood that preparations should meet sterility,pyrogenicity, general safety, and purity standards as required by theFDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed., 1990, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the pharmaceutical compositions is contemplated.

Certain embodiments of the present invention may comprise differenttypes of carriers depending on whether it is to be administered insolid, liquid, or aerosol form, and whether it needs to be sterile forthe route of administration, such as injection. The compositions can beadministered intravenously, intradermally, transdermally, intrathecally,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, intramuscularly, subcutaneously, mucosally, orally,topically, locally, by inhalation (e.g., aerosol inhalation), byinjection, by infusion, by continuous infusion, by localized perfusionbathing target cells directly, via a catheter, via a lavage, in lipidcompositions (e.g., liposomes), or by other methods or any combinationof the forgoing as would be known to one of ordinary skill in the art(see, for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990,incorporated herein by reference).

The modified polypeptides may be formulated into a composition in a freebase, neutral, or salt form. Pharmaceutically acceptable salts includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganicacids, such as, for example, hydrochloric or phosphoric acids, or suchorganic acids as acetic, oxalic, tartaric, or mandelic acid. Saltsformed with the free carboxyl groups can also be derived from inorganicbases, such as, for example, sodium, potassium, ammonium, calcium, orferric hydroxides; or such organic bases as isopropylamine,trimethylamine, histidine, or procaine. Upon formulation, solutions willbe administered in a manner compatible with the dosage formulation andin such amount as is therapeutically effective. The formulations areeasily administered in a variety of dosage forms, such as formulated forparenteral administrations, such as injectable solutions, or aerosolsfor delivery to the lungs, or formulated for alimentary administrations,such as drug release capsules and the like.

Further in accordance with certain aspects of the present invention, thecomposition suitable for administration may be provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent, or carrier is detrimental to the recipient or to thetherapeutic effectiveness of the composition contained therein, its usein administrable composition for use in practicing the methods isappropriate. Examples of carriers or diluents include fats, oils, water,saline solutions, lipids, liposomes, resins, binders, fillers, and thelike, or combinations thereof. The composition may also comprise variousantioxidants to retard oxidation of one or more component. Additionally,the prevention of the action of microorganisms can be brought about bypreservatives, such as various antibacterial and antifungal agents,including but not limited to parabens (e.g., methylparabens,propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal orcombinations thereof.

In accordance with certain aspects of the present invention, thecomposition is combined with the carrier in any convenient and practicalmanner, i.e., by solution, suspension, emulsification, admixture,encapsulation, absorption, and the like. Such procedures are routine forthose skilled in the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner, such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in acomposition include buffers, amino acids, such as glycine and lysine,carbohydrates, such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle composition that includes an engineeredmethioninase, one or more lipids, and an aqueous solvent. As usedherein, the term “lipid” will be defined to include any of a broad rangeof substances that is characteristically insoluble in water andextractable with an organic solvent. This broad class of compounds iswell known to those of skill in the art, and as the term “lipid” is usedherein, it is not limited to any particular structure. Examples includecompounds that contain long-chain aliphatic hydrocarbons and theirderivatives. A lipid may be naturally occurring or synthetic (i.e.,designed or produced by man). However, a lipid is usually a biologicalsubstance. Biological lipids are well known in the art, and include forexample, neutral fats, phospholipids, phosphoglycerides, steroids,terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides,lipids with ether- and ester-linked fatty acids, polymerizable lipids,and combinations thereof. Of course, compounds other than thosespecifically described herein that are understood by one of skill in theart as lipids are also encompassed by the compositions and methods.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the engineered methioninase or a fusion proteinthereof may be dispersed in a solution containing a lipid, dissolvedwith a lipid, emulsified with a lipid, mixed with a lipid, combined witha lipid, covalently bonded to a lipid, contained as a suspension in alipid, contained or complexed with a micelle or liposome, or otherwiseassociated with a lipid or lipid structure by any means known to thoseof ordinary skill in the art. The dispersion may or may not result inthe formation of liposomes.

The actual dosage amount of a composition administered to an animalpatient can be determined by physical and physiological factors, such asbody weight, severity of condition, the type of disease being treated,previous or concurrent therapeutic interventions, idiopathy of thepatient, and on the route of administration. Depending upon the dosageand the route of administration, the number of administrations of apreferred dosage and/or an effective amount may vary according to theresponse of the subject. The practitioner responsible for administrationwill, in any event, determine the concentration of active ingredient(s)in a composition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared in such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors, such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations, will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 milligram/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 milligram/kg/body weightto about 100 milligram/kg/body weight, about 5 microgram/kg/body weightto about 500 milligram/kg/body weight, etc., can be administered, basedon the numbers described above.

XI. Combination Treatments

In certain embodiments, the compositions and methods of the presentembodiments involve administration of an engineered methioninase incombination with a second or additional therapy. Such therapy can beapplied in the treatment of any disease that is associated withmethionine dependency. For example, the disease may be cancer.

The methods and compositions, including combination therapies, enhancethe therapeutic or protective effect, and/or increase the therapeuticeffect of another anti-cancer or anti-hyperproliferative therapy.Therapeutic and prophylactic methods and compositions can be provided ina combined amount effective to achieve the desired effect, such as thekilling of a cancer cell and/or the inhibition of cellularhyperproliferation. This process may involve contacting the cells withboth an engineered methioninase and a second therapy. A tissue, tumor,or cell can be contacted with one or more compositions orpharmacological formulation(s) comprising one or more of the agents(i.e., an engineered methioninase or an anti-cancer agent), or bycontacting the tissue, tumor, and/or cell with two or more distinctcompositions or formulations, wherein one composition provides 1) anengineered methioninase, 2) an anti-cancer agent, or 3) both anengineered methioninase and an anti-cancer agent. Also, it iscontemplated that such a combination therapy can be used in conjunctionwith chemotherapy, radiotherapy, surgical therapy, or immunotherapy.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing, for example, both agents are delivered to a cellin a combined amount effective to kill the cell or prevent it fromdividing.

An engineered methioninase may be administered before, during, after, orin various combinations relative to an anti-cancer treatment. Theadministrations may be in intervals ranging from concurrently to minutesto days to weeks. In embodiments where the engineered methioninase isprovided to a patient separately from an anti-cancer agent, one wouldgenerally ensure that a significant period of time did not expirebetween the time of each delivery, such that the two compounds wouldstill be able to exert an advantageously combined effect on the patient.In such instances, it is contemplated that one may provide a patientwith the engineered methioninase and the anti-cancer therapy withinabout 12 to 24 or 72 h of each other and, more particularly, withinabout 6-12 h of each other. In some situations it may be desirable toextend the time period for treatment significantly where several days(2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapsebetween respective administrations.

In certain embodiments, a course of treatment will last 1-90 days ormore (this such range includes intervening days). It is contemplatedthat one agent may be given on any day of day 1 to day 90 (this suchrange includes intervening days) or any combination thereof, and anotheragent is given on any day of day 1 to day 90 (this such range includesintervening days) or any combination thereof. Within a single day(24-hour period), the patient may be given one or multipleadministrations of the agent(s). Moreover, after a course of treatment,it is contemplated that there is a period of time at which noanti-cancer treatment is administered. This time period may last 1-7days, and/or 1-5 weeks, and/or 1-12 months or more (this such rangeincludes intervening days), depending on the condition of the patient,such as their prognosis, strength, health, etc. It is expected that thetreatment cycles would be repeated as necessary.

Various combinations may be employed. For the example below anengineered methioninase is “A” and an anti-cancer therapy is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of any compound or therapy of the present embodiments toa patient will follow general protocols for the administration of suchcompounds, taking into account the toxicity, if any, of the agents.Therefore, in some embodiments there is a step of monitoring toxicitythat is attributable to combination therapy.

A. Chemotherapy

A wide variety of chemotherapeutic agents may be used in accordance withthe present embodiments. The term “chemotherapy” refers to the use ofdrugs to treat cancer. A “chemotherapeutic agent” is used to connote acompound or composition that is administered in the treatment of cancer.These agents or drugs are categorized by their mode of activity within acell, for example, whether and at what stage they affect the cell cycle.Alternatively, an agent may be characterized based on its ability todirectly cross-link DNA, to intercalate into DNA, or to inducechromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents, such asthiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan,improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines, includingaltretamine, triethylenemelamine, tri etylenephosphoramide,triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, and uracil mustard;nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics, such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gammall andcalicheamicin omegall); dynemicin, including dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycini s, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; anti-metabolites, such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogues, such asdenopterin, pteropterin, and trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs, such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens, such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone; anti-adrenals, such as mitotane andtrilostane; folic acid replenisher, such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharidecomplex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′, 2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g.,paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine;platinum coordination complexes, such as cisplatin, oxaliplatin, andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan(e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids, such as retinoic acid;capecitabine; carboplatin, procarbazine,plicomycin, gemcitabien,navelbine, farnesyl-protein tansferase inhibitors, transplatinum, andpharmaceutically acceptable salts, acids, or derivatives of any of theabove.

B. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated, such as microwaves, proton beamirradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), andUV-irradiation. It is most likely that all of these factors affect abroad range of damage on DNA, on the precursors of DNA, on thereplication and repair of DNA, and on the assembly and maintenance ofchromosomes. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 wk), to single dosesof 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely,and depend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the neoplastic cells.

C. Immunotherapy

The skilled artisan will understand that immunotherapies may be used incombination or in conjunction with methods of the embodiments. In thecontext of cancer treatment, immunotherapeutics, generally, rely on theuse of immune effector cells and molecules to target and destroy cancercells. Rituximab (RITUXAN®) is such an example. The immune effector maybe, for example, an antibody specific for some marker on the surface ofa tumor cell. The antibody alone may serve as an effector of therapy orit may recruit other cells to actually affect cell killing. The antibodyalso may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

In one aspect of immunotherapy, the tumor cell must bear some markerthat is amenable to targeting, i.e., is not present on the majority ofother cells. Many tumor markers exist and any of these may be suitablefor targeting in the context of the present embodiments. Common tumormarkers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68,TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor,erb B, and p155. An alternative aspect of immunotherapy is to combineanticancer effects with immune stimulatory effects. Immune stimulatingmolecules also exist including: cytokines, such as IL-2, IL-4, IL-12,GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growthfactors, such as FLT3 ligand.

Examples of immunotherapies currently under investigation or in use areimmune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum,dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998);cytokine therapy, e.g., interferons α, β, and β, IL-1, GM-CSF, and TNF(Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998);gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998;Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-gangliosideGM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Pat.No. 5,824,311). It is contemplated that one or more anti-cancertherapies may be employed with the antibody therapies described herein.

D. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative, andpalliative surgery. Curative surgery includes resection in which all orpart of cancerous tissue is physically removed, excised, and/ordestroyed and may be used in conjunction with other therapies, such asthe treatment of the present embodiments, chemotherapy, radiotherapy,hormonal therapy, gene therapy, immunotherapy, and/or alternativetherapies. Tumor resection refers to physical removal of at least partof a tumor. In addition to tumor resection, treatment by surgeryincludes laser surgery, cryosurgery, electrosurgery, andmicroscopically-controlled surgery (Mohs' surgery).

Upon excision of part or all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection, or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

E. Other Agents

It is contemplated that other agents may be used in combination withcertain aspects of the present embodiments to improve the therapeuticefficacy of treatment. These additional agents include agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion,agents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers, or other biological agents. Increases inintercellular signaling by elevating the number of GAP junctions wouldincrease the anti-hyperproliferative effects on the neighboringhyperproliferative cell population. In other embodiments, cytostatic ordifferentiation agents can be used in combination with certain aspectsof the present embodiments to improve the anti-hyperproliferativeefficacy of the treatments. Inhibitors of cell adhesion are contemplatedto improve the efficacy of the present embodiments. Examples of celladhesion inhibitors are focal adhesion kinase (FAKs) inhibitors andLovastatin. It is further contemplated that other agents that increasethe sensitivity of a hyperproliferative cell to apoptosis, such as theantibody c225, could be used in combination with certain aspects of thepresent embodiments to improve the treatment efficacy.

XII. Kits

Certain aspects of the present invention may provide kits, such astherapeutic kits. For example, a kit may comprise one or morepharmaceutical composition as described herein and optionallyinstructions for their use. Kits may also comprise one or more devicesfor accomplishing administration of such compositions. For example, asubject kit may comprise a pharmaceutical composition and catheter foraccomplishing direct intravenous injection of the composition into acancerous tumor. In other embodiments, a subject kit may comprisepre-filled ampoules of an engineered methioninase, optionally formulatedas a pharmaceutical, or lyophilized, for use with a delivery device.

Kits may comprise a container with a label. Suitable containers include,for example, bottles, vials, and test tubes. The containers may beformed from a variety of materials, such as glass or plastic. Thecontainer may hold a composition that includes an engineeredmethioninase that is effective for therapeutic or non-therapeuticapplications, such as described above. The label on the container mayindicate that the composition is used for a specific therapy ornon-therapeutic application, and may also indicate directions for eitherin vivo or in vitro use, such as those described above. The kit of theinvention will typically comprise the container described above and oneor more other containers comprising materials desirable from acommercial and user standpoint, including buffers, diluents, filters,needles, syringes, and package inserts with instructions for use.

XIII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Cystathionine-γ-lyase Engineered for Methionine-γ-lyaseActivity

CGL is a tetramer that catalyzes the last step in the mammaliantranssulfuration pathway (Rao et al., 1990). CGL catalyzes theconversion of L-cystathionine to L-cysteine, alpha-ketobutyrate, andammonia. The human CGL (hCGL) cDNA has previously been cloned andexpressed, but with relatively low yields (˜5 mg/L culture) (Lu et al.,1992; Steegborn et al., 1999). Using sequence and structural alignmentsof CGL and MGL enzymes as a guide, hCGL was converted to an enzyme forthe efficient degradation of methionine.

Example 2 Gene Synthesis and Expression of Improved Modified HumanCystathionine-γ-lyase

The human cystathionine-γ-lyase gene contains multiple codons that arerarely utilized in E. coli and can interfere with expression. Thus, inorder to optimize protein expression in E. coli, the respective geneswere assembled with codon optimized oligonucleotides designed usingDNA-Works software (Hoover et al., 2002). Each construct contains anN-terminal NcoI restriction site, an in-frame N-terminal His6 tag, and aC-terminal EcoRI site for simplifying cloning. After cloning into apET28a vector (Novagen), E. coli (BL21) containing an appropriatecystathionase expression vector were grown at 37° C. using TerrificBroth (TB) media containing 50 μg/mL kanamycin in shaker flasks at 250rpm until reaching an OD₆₀₀ of ˜0.5-0.6. At this point the cultures wereswitched to a shaker at 25° C., induced with 0.5 mM IPTG, and allowed toexpress protein for an additional 12 h. Cell pellets were then collectedby centrifugation and re-suspended in an IMAC buffer (10 mM NaPO₄/10 mMimidazole/300 mM NaCl, pH 8). After lysis by a French pressure cell,lysates were centrifuged at 20,000×g for 20 min at 4° C., and theresulting supernatant applied to a nickel IMAC column, washed with 10-20column volumes of IMAC buffer, and then eluted with an IMAC elutionbuffer (50 mM NaPO₄/250 mM imidazole/300 mM NaCl, pH 8). Fractionscontaining enzyme were then incubated with 10 mM pyridoxal-5′-phosphate(PLP) for an hour at 25° C. Using a 10,000 MWCO centrifugal filterdevice (AMICON®), proteins were then buffer exchanged several times intoa 100 mM PBS, 10% glycerol, pH 7.3 solution. Enzyme aliquots were thenflash frozen in liquid nitrogen and stored at −80° C. CGL and CGLvariants purified in this manner were >95% homogeneous as assessed bySDS-PAGE and coomassie staining. The yield was calculated to be ˜400mg/L culture based upon the calculated extinction coefficient,λ₂₈₀=29,870 M⁻¹cm⁻¹ in a final buffer concentration of 6 M guanidiniumhydrochloride, 20 mM phosphate buffer, pH 6.5 (Gill and von Hippel,1989).

Example 3 96-well Plate Screen for Methionine-γ-lyase Activity andRanking Clones

Both MGL and CGL produce 2-ketobutanoic acid from their respectivesubstrates. A colorimetric assay for the detection of α-keto acids using3-methylbenzothiazolin-2-one hydrazone (MBTH) (Takakura et al., 2004)was scaled to a 96-well plate format for screening small libraries andfor ranking clones with the greatest METase (methionine-γ-lyase)activity. This plate screen provides a facile method for picking themost active clones from the mutagenic libraries. Clones displayinggreater activity than parental controls are selected for furthercharacterization, thus eliminating the need to purify more than a fewvariants for kinetic analysis.

Single colonies containing mutagenized hCGL, hCGL or pMGL were pickedinto 96-well culture plates containing 75 μL of TB media/well containing50 μg/mL kanamycin. These cultures were then grown at 37° C. on a plateshaker until reaching an OD₆₀₀ of ˜0.8-1. After cooling to 25° C., anadditional 75 μL of media/well containing 50 μg/mL kanamycin and 0.5 mMIPTG was added. Expression was performed at 25° C. with shaking for 2 h,following which 100 μL of culture/well was transferred to a 96-wellassay plate. The assay plates were then centrifuged to pellet the cells,the media was removed, and the cells were lysed by addition of 50μL/well of B-PER® protein extraction reagent (Pierce). After clearing bycentrifugation, the lysate was incubated with 5 mM L-Met at 37° C. for10-12 h. The reaction was then derivatized by addition of 3 parts of0.03% MBTH solution in 1 M sodium acetate, pH 5. The plates were heatedat 50° C. for 40 min and after cooling were read at 320 nm in amicrotiter plate reader.

Example 4 Library Design for Generating Improved Methionine DegradingVariants Derived from hCGL-E59N-R119L-E339V

The gene for the hCGL-E59N-R119L-E339V (hCGL-NLV) methionine degradingenzyme was used as a starting point to generate further variants withimprovements in activity. Amino acid sequence alignments were generatedusing MGL and CGL sequences from various organisms. Regions selected formutagenesis were identified at specific alignment sites where the MGLenzymes all had one conserved residue and the CGL enzymes all had adifferent conserved residue. Although MGL and CGL have highly homologousstructures they do not degrade each other's respective substrate, thusphylogenetically conserved amino acid sequence differences between MGLand CGL enzymes can indicate residues that are important for degradingtheir respective substrate. Libraries were generated by overlapextension PCR using oligonucleotides containing either a codon for theparent hCGL-NLV template coding for a conserved residue or a codon forthe corresponding MGL conserved residue. The final assembled PCRproducts were digested with NcoI and coRI and ligated into pET28a vectorwith T4 DNA ligase. The resulting ligations were transformed directlyinto E. coli (BL21) and plated on LB-kanamycin plates for subsequentscreening as described in Example 3. Two times more colonies than thetheoretical diversity of the libraries (i.e., all possible genesequences encoded by the library) were screened. Clones displayinggreater activity than the parent hCGL-NLV variant were isolated andsequenced to identify the mutations conferring improved activity. Cloneswith improved L-methionine degrading activity were used as templates initerative rounds of mutagenesis as described.

Example 5 Characterization of Improved Human Methionine DegradingVariants

After several rounds of mutagenesis and screening as described inExamples 3 and 4, five amino acid positions in addition to the originalthree mutation sites (i.e. E59N-R119L-E339V) were found to conferimproved methionine degrading activity compared to hCGL-NLV (SEQ ID NO:2). These additional positions are located at hCGL (SEQ ID NO: 1)residues 63, 91, 268, 311, and 353 (see, FIG. 1). Mutation of one ormore of these positions to S63L, L91M, K268R, T311G, and I353S, incombination with mutations of residues at positions 59, 119, and 339,resulted in improved k_(cat)/K_(M) values for degrading L-methioninecompared to hCGL-NLV. In particular, the variants containing amino acidsubstitutions corresponding to SEQ ID NO: 3,hCGL-E59N-S63L-L91M-R119L-K268R-T311G-E339V-I353S (hCGL-8mut-1); SEQ IDNO: 4, hCGL-E59I-S63L-L91M-R119L-K268R-T311G-E339V-I353S (hCGL-8mut-2);SEQ ID NO: 5, hCGL-E59N-S63L-L91M-R119A-K268R-T311G-E339V-I353S(hCGL-8mut-3); and SEQ ID NO: 6,hCGL-E59I-S63L-L91M-R119A-K268R-T311G-E339V-I353S (hCGL-8mut-4) wereshown to have the highest k_(cat)/K_(M) values for degradingL-methionine. These variants (hCGL-8mut-(1-4)) were purified to greaterthan 95% homogeneity as assessed by SDS-PAGE as described in Example 2and kinetically characterized for their ability to degrade L-Met in a100 mM PBS buffer at pH 7.3 and 37° C. using a 1 mL scale MBTH assaysimilar to that described in Example 3.

TABLE 1 Comparison of Michaelis-Menten kinetics of L-methioninedegradation at pH 7.3 and 37° C. using hCGL, hCGL-NLV, and improvedvariants hCGL-8mut(1-4). Variant k_(cat) s⁻¹ K_(M) mM k_(cat)/K_(M)s⁻¹M⁻¹ hCGL 0 0 0 hCGL-NLV 7.9 14 560 hCGL-8mut-1 7.9 2.2 3590hCGL-8mut-2 7.1 1.4 5070 hCGL-8mut-3 ND ND ND hCGL-8mut-4 9.8 1.8 5440ND = not determined

Example 6 Cytotoxicity of hCGL-8mut-1 Against Tumor Cell Lines

The in vitro cytotoxicity of hCGL-8mut-1 was assessed against melanomacell line A375 and prostate cancer cell lines DU145 and PC3. Cells wereseeded at 3000 cells/well in 96-well culture plates in DMEM media forthe A375 cells or RPMI-1640 media for the prostate tumor cell lines andallowed to grow for 24 h before treatment with varying concentrations ofenzyme. After 5 days of treatment, proliferation was measured using the(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay. Analysis of the resulting data for A375 yielded an apparentIC₅₀ value of 0.08 μM and apparent IC₅₀ values of 0.21 μM for DU145 and0.25 μM for PC3 prostate tumor cells (FIG. 2).

Example 7 Pharmacological Preparation of hCGL-8mut-1

The hCGL-8mut-1 enzyme was purified as described in Example 2 with oneexception: after binding to the IMAC column, the protein was washedextensively (90-100 column volumes) with an IMAC buffer containing 0.1%TRITON® 114. Then the column was washed with 10-20 column volumes ofIMAC buffer and eluted with an IMAC elution buffer (50 mM NaPO₄/250 mMimidazole/300 mM NaCl, pH 8). Washing with TRITON® 114 was employed forendotoxin removal. The purified protein was subjected to buffer exchangeinto a 100 mM NaPO4 buffer at pH 8.3 using a 10,000 MWCO filtrationdevice (Amicon). Subsequently, PLP was added at a concentration of 10 mMand the protein was incubated for 1 h at 25° C. Methoxy PEG SuccinimidylCarboxymethyl Ester 5000 MW (JenKem Technology) was then added tohCGL-8mut-1 at an 80:1 molar ratio and allowed to react for 1 h at 25°C. under constant stirring. The resulting mixture was extensively bufferexchanged (PBS with 10% glycerol) using a 100,000 MWCO filtration device(Amicon), and sterilized with a 0.2 micron syringe filter (VWR). AllPEGylated enzymes were analyzed for lipopolysaccharide (LPS) contentusing a Limulus Amebocyte Lysate (LAL) kit (Cape Cod Incorporated).

Example 8 Serum Stability of PEGylated hCGL-8mut-1

The serum stability of PEGylated hCGL-8mut-1 was tested by incubation ofthe enzyme in pooled human serum at 37° C. at a final concentration of10 μM. At different time points, aliquots were withdrawn and tested foractivity using the DTNB assay as described in U.S. Pat. Publ.2011/0200576, which is incorporated herein by reference in its entirety.After plotting the data, PEGylated hCGL-8mut-1 was calculated to have ahalf-life (T_(0.5)) of 101±4 h (FIG. 3).

Example 9 Pharmacodymanic Analysis of PEGylated hCGL-8mut-1 in Mice

To assess the efficacy of the engineered human methionine degradingenzymes disclosed in Examples 4-7 above in clearing L-methionine in amouse model, three groups of three animals each were administered 50mg/kg of PEG-hCGL-8mut-1 by tail vein injection, while being maintainedon a normal diet. Groups were sacrificed at time points corresponding to8, 24, and 48 h by cardiac venipuncture for serum collection. The serumsamples were then analyzed for L-methionine content by derivatizationwith o-phthalaldehyde (OPA) followed by high performance liquidchromatography (HPLC) essentially as described by Agilent Technologies(on the world wide web atchem.agilent.com/Library/datasheets/Public/5980-3088.PDF). This dosingscheme using PEG-hCGL-8mut-1 enabled depletion of L-methionine to levelslower than 5 μM for over 15 h (FIG. 4). In contrast, administration of200 mg/kg of the PEGylated hCGL-NLV to mice on a normal diet onlylowered serum L-methionine to ˜10 μM for 4 h (FIG. 5).

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A polypeptide comprising a variant of a nativeprimate cystathionine-γ-lyase (CGL) enzyme, wherein the variant CGL hasmethionine-γ-lyase activity, comprises a sequence at least 95% identicalto SEQ ID NO: 1, and comprises amino acid substitutions comprising E59N,R119L, and E339V of SEQ ID NO: 1, and at least one of the followingsubstitutions: S63L, L91M, K268R, T311G, and I353S.
 2. The polypeptideof claim 1, wherein the variant CGL has at least 98% sequence identityto SEQ ID NO:
 1. 3. The polypeptide of claim 1, wherein the variant CGLis human CGL comprising SEQ ID NO: 1 having the E59N, S63L, L91M, R119L,K268R, T311G, E339V, and I353S substitutions.
 4. The polypeptide ofclaim 1, further comprising a heterologous peptide segment.
 5. Thepolypeptide of claim 4, wherein the heterologous peptide segment is anXTEN peptide, an IgG Fc, an albumin, or an albumin binding peptide. 6.The polypeptide of claim 1, wherein the polypeptide is coupled topolyethylene glycol (PEG).
 7. The polypeptide of claim 6, wherein thepolypeptide is coupled to PEG via one or more Lys or Cys residues.
 8. Apharmaceutical formulation comprising the polypeptide of claim 1 in apharmaceutically acceptable carrier.
 9. A method of treating a tumorcell or subject having a tumor cell comprising administering to thetumor cell or the subject the formulation of claim
 8. 10. The method ofclaim 9, wherein the subject is maintained on a methionine restricteddiet.
 11. The method of claim 9, wherein the subject is maintained on anormal diet.
 12. The method of claim 9, wherein the subject is a humanpatient.
 13. The method of claim 9, wherein the formulation isadministered intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostaticaly, intrapleurally, intratracheally, intraocularly,intranasally, intravitreally, intravaginally, intrarectally,intramuscularly, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, orally, by inhalation,by injection, by infusion, by continuous infusion, by localizedperfusion bathing target cells directly, via a catheter, or via alavage.
 14. The method of claim 9, wherein the formulation isadministered to a nutrient medium of the tumor cell.
 15. The method ofclaim 14, wherein the nutrient medium is blood, lymphatic fluid, orspinal fluid.
 16. The method of claim 9, further comprisingadministering at least a second anticancer therapy to the subject. 17.The method of claim 16, wherein the second anticancer therapy is asurgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonetherapy, immunotherapy or cytokine therapy.
 18. The method of claim 9,wherein the tumor cell is a cell of breast cancer, prostate cancer,neuroblastoma, or pancreatic carcinoma.